Compositions and methods for siRNA inhibition of angiogenesis

ABSTRACT

RNA interference using small interfering RNAs which are specific for the vascular endothelial growth factor (VEGF) gene and the VEGF receptor genes Flt-1 and Flk-1/KDR inhibit expression of these genes. Diseases which involve angiogenesis stimulated by overexpression of VEGF, such as diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering the small interfering RNAs.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional patentapplication serial No. 60/398,417, filed on Jul. 24, 2002.

REFERENCE TO GOVERNMENT GRANT

[0002] The invention described herein was supported in part by NIH/NEIgrant no. R01-EY10820. The U.S. government has certain rights in thisinvention.

FIELD OF THE INVENTION

[0003] This invention relates to the regulation of gene expression bysmall interfering RNA, in particular for treating diseases or conditionsinvolving angiogenesis.

BACKGROUND OF THE INVENTION

[0004] Angiogenesis, defined as the growth of new capillary bloodvessels or “neovascularization,” plays a fundamental role in growth anddevelopment. In mature humans, the ability to initiate angiogenesis ispresent in all tissues, but is held under strict control. A keyregulator of angiogenesis is vascular endothelial growth factor(“VEGF”), also called vascular permeability factor (“VPF”). VEGF existsin at least four different alternative splice forms in humans (VEGF₁₂₁,VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆), all of which exert similar biologicalactivities.

[0005] Angiogenesis is initiated when secreted VEGF binds to the Flt-1and Flk-1/KDR receptors (also called VEGF receptor 1 and VEGF receptor2), which are expressed on the surface of endothelial cells. Flt-1 andFlk-1/KDR are transmembrane protein tyrosine kinases, and binding ofVEGF initiates a cell signal cascade resulting in the ultimateneovascularization in the surrounding tissue.

[0006] Aberrant angiogenesis, or the pathogenic growth of new bloodvessels, is implicated in a number of conditions. Among these conditionsare diabetic retinopathy, psoriasis, exudative or “wet” age-relatedmacular degeneration (“ARMD”), rheumatoid arthritis and otherinflammatory diseases, and most cancers. The diseased tissues or tumorsassociated with these conditions express abnormally high levels of VEGF,and show a high degree of vascularization or vascular permeability.

[0007] ARMD in particular is a clinically important angiogenic disease.This condition is characterized by choroidal neovascularization in oneor both eyes in aging individuals, and is the major cause of blindnessin industrialized countries.

[0008] A number of therapeutic strategies exist for inhibiting aberrantangiogenesis, which attempt to reduce the production or effect of VEGF.For example, anti-VEGF or VEGF receptor antibodies (Kim ES et al.(2002), PNAS USA 99: 11399-11404), and soluble VEGF “traps” whichcompete with endothelial cell receptors for VEGF binding (Holash J etal. (2002), PNAS USA 99: 11393-11398) have been developed. ClassicalVEGF “antisense” or aptamer therapies directed against VEGF geneexpression have also been proposed (U.S. published application2001/0021772 of Uhlmann et al.). However, the anti-angiogenic agentsused in these therapies can produce only a stoichiometric reduction inVEGF or VEGF receptor, and the agents are typically overwhelmed by theabnormally high production of VEGF by the diseased tissue. The resultsachieved with available anti-angiogenic therapies have therefore beenunsatisfactory.

[0009] RNA interference (hereinafter “RNAi”) is a method ofpost-transcriptional gene regulation that is conserved throughout manyeukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide)double stranded RNA (“dsRNA”) molecules which are present in the cell(Fire A et al. (1998), Nature 391: 806-811). These short dsRNAmolecules, called “short interfering RNA” or “siRNA,” cause thedestruction of messenger RNAs (“mRNAs”) which share sequence homologywith the siRNA to within one nucleotide resolution (Elbashir S M et al.(2001), Genes Dev, 15: 188-200). It is believed that the siRNA and thetargeted mRNA bind to an “RNA-induced silencing complex” or “RISC”,which cleaves the targeted mRNA. The siRNA is apparently recycled muchlike a multiple-turnover enzyme, with 1 siRNA molecule capable ofinducing cleavage of approximately 1000 mRNA molecules. siRNA-mediatedRNAi degradation of an mRNA is therefore more effective than currentlyavailable technologies for inhibiting expression of a target gene.

[0010] Elbashir S M et al. (2001), supra, has shown that synthetic siRNAof 21 and 22 nucleotides in length, and which have short 3′ overhangs,are able to induce RNAi of target mRNA in a Drosophila cell lysate.Cultured mammalian cells also exhibit RNAi degradation with syntheticsiRNA (Elbashir S M et al. (2001) Nature, 411: 494-498), and RNAidegradation induced by synthetic siRNA has recently been shown in livingmice (McCaffrey AP et al. (2002), Nature, 418: 38-39; Xia H et al.(2002), Nat. Biotech. 20: 1006-1010). The therapeutic potential ofsiRNA-induced RNAi degradation has been demonstrated in several recentin vitro studies, including the siRNA-directed inhibition of HIV-1infection (Novina C D et al. (2002), Nat. Med. 8: 681-686) and reductionof neurotoxic polyglutamine disease protein expression (Xia H et al.(2002), supra).

[0011] What is needed, therefore, are agents which selectively inhibitexpression of VEGF or VEGF receptors in catalytic or sub-stoichiometricamounts.

SUMMARY OF THE INVENTION

[0012] The present invention is directed to siRNAs which specificallytarget and cause RNAi-induced degradation of mRNA from VEGF, Flt-1 andFlk-1/KDR genes. The siRNA compounds and compositions of the inventionare used to inhibit angiogenesis, in particular for the treatment ofcancerous tumors, age-related macular degeneration, and other angiogenicdiseases.

[0013] Thus, the invention provides an isolated siRNA which targetshuman VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA, or analternative splice form, mutant or cognate thereof. The siRNA comprisesa sense RNA strand and an antisense RNA strand which form an RNA duplex.The sense RNA strand comprises a nucleotide sequence identical to atarget sequence of about 19 to about 25 contiguous nucleotides in thetarget mRNA.

[0014] The invention also provides recombinant plasmids and viralvectors which express the siRNA of the invention, as well aspharmaceutical compositions comprising the siRNA of the invention and apharmaceutically acceptable carrier.

[0015] The invention further provides a method of inhibiting expressionof human VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA, or analternative splice form, mutant or cognate thereof, comprisingadministering to a subject an effective amount of the siRNA of theinvention such target mRNA is degraded.

[0016] The invention further provides a method of inhibitingangiogenesis in a subject, comprising administering to a subject aneffective amount of an siRNA targeted to human VEGF mRNA, human Flt-1mRNA, human Flk-1/KDR mRNA, or an alternative splice form, mutant orcognate thereof.

[0017] The invention further provides a method of treating an angiogenicdisease, comprising administering to a subject in need of such treatmentan effective amount of an siRNA targeted to human VEGF mRNA, human Flt-1mRNA, human Flk-1/KDR mRNA, or an alternative splice form, mutant orcognate thereof, such that angiogenesis associated with the angiogenicdisease is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIGS. 1A and 1B are a histograms of VEGF concentration (in pg/ml)in hypoxic 293 and HeLa cells treated with no siRNA (“−”); nonspecificsiRNA (“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). VEGFconcentration (in pg/ml) in non-hypoxic 293 and HeLa cells is alsoshown. Each bar represents the average of four experiments, and theerror is the standard deviation of the mean.

[0019]FIG. 2 is a histogram of murine VEGF concentration (in pg/ml) inhypoxic NIH 3T3 cells treated with no siRNA (“−”); nonspecific siRNA(“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). Each barrepresents the average of six experiments and the error is the standarddeviation of the mean.

[0020]FIG. 3 is a histogram of human VEGF concentration (pg/totalprotein) in retinas from mice injected with adenovirus expressing humanVEGF (“AdVEGF”) in the presence of either GFP siRNA (dark gray bar) orhuman VEGF siRNA (light grey bar). Each bar represent the average of 5eyes and the error bars represent the standard error of the mean(S.E.M.).

[0021]FIG. 4 is a histogram showing the mean area (in mm²) oflaser-induced CNV in control eyes given subretinal injections of GFPsiRNA (N=9; “GFP siRNA”), and in eyes given subretinal injections ofmouse VEGF siRNA (N=7; “Mouse VEGF siRNA”). The error bars represent thestandard error of the mean (S.E.M.).

[0022]FIG. 5 is a schematic representation of pAAVsiRNA, a cis-actingplasmid used to generate a recombinant AAV viral vector of theinvention. “ITR”: AAV inverted terminal repeats; “U6”: U6 RNA promoters;“Sense”: siRNA sense coding sequence; “Anti”: siRNA antisense codingsequence; “PolyT”: polythymidine termination signals.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Unless otherwise indicated, all nucleic acid sequences herein aregiven in the 5′ to 3′ direction. Also, all deoxyribonucleotides in anucleic acid sequence are represented by capital letters (e.g.,deoxythymidine is “T”), and ribonucleotides in a nucleic acid sequenceare represented by lower case letters (e.g., uridine is “u”).

[0024] Compositions and methods comprising siRNA targeted to VEGF, Flt-1or Flk-1/KDR mRNA are advantageously used to inhibit angiogenesis, inparticular for the treatment of angiogenic disease. The siRNA of theinvention are believed to cause the RNAi-mediated degradation of thesemRNAs, so that the protein product of the VEGF, Flt-1 or Flk-1/KDR genesis not produced or is produced in reduced amounts. Because VEGF bindingto the Flt-1 or Flk-1/KDR receptors is required for initiating andmaintaining angiogenesis, the siRNA-mediated degradation of VEGF, Flt-1or Flk-1/KDR mRNA inhibits the angiogenic process.

[0025] The invention therefore provides isolated siRNA comprising shortdouble-stranded RNA from about 17 nucleotides to about 29 nucleotides inlength, preferably from about 19 to about 25 nucleotides in length, thatare targeted to the target mRNA. The siRNA comprise a sense RNA strandand a complementary antisense RNA strand annealed together by standardWatson-Crick base-pairing interactions (hereinafter “base-paired”). Asis described in more detail below, the sense strand comprises a nucleicacid sequence which is identical to a target sequence contained withinthe target mRNA.

[0026] The sense and antisense strands of the present siRNA can comprisetwo complementary, single-stranded RNA molecules or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area. Withoutwishing to be bound by any theory, it is believed that the hairpin areaof the latter type of siRNA molecule is cleaved intracellularly by the“Dicer” protein (or its equivalent) to form an siRNA of two individualbase-paired RNA molecules (see Tuschl, T. (2002), supra).

[0027] As used herein, “isolated” means altered or removed from thenatural state through human intervention. For example, an siRNAnaturally present in a living animal is not “isolated,” but a syntheticsiRNA, or an siRNA partially or completely separated from the coexistingmaterials of its natural state is “isolated.” An isolated siRNA canexist in substantially purified form, or can exist in a non-nativeenvironment such as, for example, a cell into which the siRNA has beendelivered.

[0028] As used herein, “target mRNA” means human VEGF, Flt-1 orFlk-1/KDR mRNA, mutant or alternative splice forms of human VEGF, Flt-1or Flk-1/KDR mRNA, or mRNA from cognate VEGF, Flt-1 or Flk-1/KDR genes.

[0029] As used herein, a gene or mRNA which is “cognate” to human VEGF,Flt-1 or Flk-1/KDR is a gene or mRNA from another mammalian specieswhich is homologous to human VEGF, Flt-1 or Flk-1/KDR. For example, thecognate VEGF mRNA from the mouse is given in SEQ ID NO: 1.

[0030] Splice variants of human VEGF are known, including VEGF₁₂₁ (SEQID NO: 2), VEGF₁₆₅ (SEQ ID NO: 3), VEGF₁₈₉ (SEQ ID NO: 4) and VEGF₂₀₆(SEQ ID NO: 5). The mRNA transcribed from the human VEGF, Flt-1 (SEQ IDNO: 6) or Flk-1/KDR (SEQ ID NO: 7) genes can be analyzed for furtheralternative splice forms using techniques well-known in the art. Suchtechniques include reverse transcription-polymerase chain reaction(RT-PCR), northern blotting and in-situ hybridization. Techniques foranalyzing mRNA sequences are described, for example, in Busting SA(2000), J. Mol. Endocrinol. 25: 169-193, the entire disclosure of whichis herein incorporated by reference. Representative techniques foridentifying alternatively spliced mRNAs are also described below.

[0031] For example, databases that contain nucleotide sequences relatedto a given disease gene can be used to identify alternatively splicedmRNA. Such databases include GenBank, Embase, and the Cancer GenomeAnatomy Project (CGAP) database. The CGAP database, for example,contains expressed sequence tags (ESTs) from various types of humancancers. An mRNA or gene sequence from the VEGF, Flt-1 or Flk-1/KDRgenes can be used to query such a database to determine whether ESTsrepresenting alternatively spliced mRNAs have been found for a thesegenes.

[0032] A technique called “RNAse protection” can also be used toidentify alternatively spliced VEGF, Flt-1 or Flk-1/KDR mRNAs. RNAseprotection involves translation of a gene sequence into synthetic RNA,which is hybridized to RNA derived from other cells; for example, cellsfrom tissue at or near the site of neovascularization. The hybridizedRNA is then incubated with enzymes that recognize RNA:RNA hybridmismatches. Smaller than expected fragments indicate the presence ofalternatively spliced mRNAs. The putative alternatively spliced mRNAscan be cloned and sequenced by methods well known to those skilled inthe art.

[0033] RT-PCR can also be used to identify alternatively spliced VEGF,Flt-1 or Flk-1/KDR mRNAs. In RT-PCR, mRNA from the diseased tissue isconverted into cDNA by the enzyme reverse transcriptase, using methodswell-known to those of ordinary skill in the art. The entire codingsequence of the cDNA is then amplified via PCR using a forward primerlocated in the 3′ untranslated region, and a reverse primer located inthe 5′ untranslated region. The amplified products can be analyzed foralternative splice forms, for example by comparing the size of theamplified products with the size of the expected product from normallyspliced mRNA, e.g., by agarose gel electrophoresis. Any change in thesize of the amplified product can indicate alternative splicing.

[0034] mRNA produced from mutant VEGF, Flt-1 or Flk-1/KDR genes can alsobe readily identified through the techniques described above foridentifying alternative splice forms. As used herein, “mutant” VEGF,Flt-1 or Flk-1/KDR genes or mRNA include human VEGF, Flt-1 or Flk-1/KDRgenes or mRNA which differ in sequence from the VEGF, Flt-1 or Flk-1/KDRsequences set forth herein. Thus, allelic forms of these genes, and themRNA produced from them, are considered “mutants” for purposes of thisinvention.

[0035] The siRNA of the invention can comprise partially purified RNA,substantially pure RNA, synthetic RNA, or recombinantly produced RNA, aswell as altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, including modifications that make the siRNAresistant to nuclease digestion.

[0036] One or both strands of the siRNA of the invention can alsocomprise a 3′ overhang. As used herein, a “3′ overhang” refers to atleast one unpaired nucleotide extending from the 3′-end of an RNAstrand.

[0037] Thus in one embodiment, the siRNA of the invention comprises atleast one 3′ overhang of from 1 to about 6 nucleotides (which includesribonucleotides or deoxynucleotides) in length, preferably from 1 toabout 5 nucleotides in length, more preferably from 1 to about 4nucleotides in length, and particularly preferably from about 2 to about4 nucleotides in length.

[0038] In the embodiment in which both strands of the siRNA moleculecomprise a 3′ overhang, the length of the overhangs can be the same ordifferent for each strand. In a most preferred embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA of the invention cancomprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid(“uu”).

[0039] In order to enhance the stability of the present siRNA, the 3′overhangs can be also stabilized against degradation. In one embodiment,the overhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′ hydroxyl in the 2′-deoxythymidinesignificantly enhances the nuclease resistance of the 3′ overhang intissue culture medium.

[0040] In certain embodiments, the siRNA of the invention comprises thesequence AA(N19)TT or NA(N21), where N is any nucleotide. These siRNAcomprise approximately 30-70% GC, and preferably comprise approximately50% G/C. The sequence of the sense siRNA strand corresponds to (N19)TTor N21 (i.e., positions 3 to 23), respectively. In the latter case, the3′ end of the sense siRNA is converted to TT. The rationale for thissequence conversion is to generate a symmetric duplex with respect tothe sequence composition of the sense and antisense strand 3′ overhangs.The antisense RNA strand is then synthesized as the complement topositions 1 to 21 of the sense strand.

[0041] Because position 1 of the 23-nt sense strand in these embodimentsis not recognized in a sequence-specific manner by the antisense strand,the 3′-most nucleotide residue of the antisense strand can be chosendeliberately. However, the penultimate nucleotide of the antisensestrand (complementary to position 2 of the 23-nt sense strand in eitherembodiment) is generally complementary to the targeted sequence.

[0042] In another embodiment, the siRNA of the invention comprises thesequence NAR(N17)YNN, where R is a purine (e.g., A or G) and Y is apyrimidine (e.g., C or U/T). The respective 21-nt sense and antisenseRNA strands of this embodiment therefore generally begin with a purinenucleotide. Such siRNA can be expressed from pol III expression vectorswithout a change in targeting site, as expression of RNAs from pol IIIpromoters is only believed to be efficient when the first transcribednucleotide is a purine.

[0043] The siRNA of the invention can be targeted to any stretch ofapproximately 19-25 contiguous nucleotides in any of the target mRNAsequences (the “target sequence”). Techniques for selecting targetsequences for siRNA are given, for example, in Tuschl T et al., “ThesiRNA User Guide,” revised Oct. 11, 2002, the entire disclosure of whichis herein incorporated by reference. “The siRNA User Guide” is availableon the world wide web at a website maintained by Dr. Thomas Tuschl,Department of Cellular Biochemistry, AG 105, Max-Planck-Institute forBiophysical Chemistry, 37077 Gottingen, Germany, and can be found byaccessing the website of the Max Planck Institute and searching with thekeyword “siRNA.” Thus, the sense strand of the present siRNA comprises anucleotide sequence identical to any contiguous stretch of about 19 toabout 25 nucleotides in the target mRNA.

[0044] Generally, a target sequence on the target mRNA can be selectedfrom a given cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′ direction) from thestart codon. The target sequence can, however, be located in the 5′ or3′ untranslated regions, or in the region nearby the start codon (see,e.g., the target sequences of SEQ ID NOS: 73 and 74 in Table 1 below,which are within 100 nt of the 5′-end of the VEGF₁₂₁ cDNA

[0045] For example, a suitable target sequence in the VEGF₁₂₀ cDNAsequence is:

[0046] TCATCACGAAGTGGTGAAG (SEQ ID NO: 8)

[0047] Thus, an siRNA of the invention targeting this sequence, andwhich has 3′ uu overhangs on each strand (overhangs shown in bold), is:5′-ucaucacgaaguggugaaguu-3′ (SEQ ID NO:9) 3′-uuaguagugcuucaccacuuc-5′(SEQ ID NO:10)

[0048] An siRNA of the invention targeting this same sequence, buthaving 3′ TT overhangs on each strand (overhangs shown in bold) is:   5′-ucaucacgaaguggugaagTT-3′ (SEQ ID NO:11)3′-TTaguagugcuucaccacuuc-5′ (SEQ ID NO:12)

[0049] Other VEGF₁₂₁ target sequences from which siRNA of the inventioncan be derived are given in Table 1. It is understood that all VEGF₁₂₁target sequences listed herein are within that portion of the VEGF₁₂₁alternative splice form which is common to all human VEGF alternativesplice forms. Thus, these target sequences can also target VEGF₁₆₅,VEGF₁₈₉ and VEGF₂₀₆ mRNA. An example of a target sequence which targetsVEGF₁₆₅ mRNA but not VEGF₁₂₁ mRNA is AACGTACTTGCAGATGTGACA (SEQ ID NO:13). TABLE 1 VEGF₁₂₁ Target Sequences SEQ ID target sequence NO:GTTCATGGATGTCTATCAG 14 TCGAGACCCTGGTGGACAT 15 TGACGAGGGCCTGGAGTGT 16TGACGAGGGCCTGGAGTGT 17 CATCACCATGCAGATTATG 18 ACCTCACCAAGGCCAGCAC 19GGCCAGCACATAGGAGAGA 20 CAAATGTGAATGCAGACCA 21 ATGTGAATGCAGACCAAAG 22TGCAGACCAAAGAAAGATA 23 AGAAAGATAGAGCAAGACA 24 GAAAGATAGAGCAAGACAA 25GATAGAGCAAGACAAGAAA 26 GACAAGAAAATCCCTGTGG 27 GAAAATCCCTGTGGGCCTT 28AATCCCTGTGGGCCTTGCT 29 TCCCTGTGGGCCTTGCTCA 30 GCATTTGTTTGTACAAGAT 31GATCCGCAGACGTGTAAAT 32 ATGTTCCTGCAAAAACACA 33 TGTTCCTGCAAAAACACAG 34AAACACAGACTCGCGTTGC 35 AACACAGACTCGCGTTGCA 36 ACACAGACTCGCGTTGCAA 37CACAGACTCGCGTTGCAAG 38 GGCGAGGCAGCTTGAGTTA 39 ACGAACGTACTTGCAGATG 40CGAACGTACTTGCAGATGT 41 CGTACTTGCAGATGTGACA 42 GTGGTCCCAGGCTGCACCC 43GGAGGAGGGCAGAATCATC 44 GTGGTGAAGTTCATGGATG 45 AATCATCACGAAGTGGTGAAG 46AAGTTCATGGATGTCTATCAG 47 AATCGAGACCCTGGTGGACAT 48 AATGACGAGGGCCTGGAGTGT49 AACATCACCATGCAGATTATG 50 AAACCTCACCAAGGCCAGCAC 51AAGGCCAGCACATAGGAGAGA 52 AACAAATGTGAATGCAGACCA 53 AAATGTGAATGCAGACCAAAG54 AATGCAGACCAAAGAAAGATA 55 AAAGAAAGATAGAGCAAGACA 56AAGAAAGATAGAGCAAGACAA 57 AAGATAGAGCAAGACAAGAAAAT 58AAGACAAGAAAATCCCTGTGGGC 59 AAGAAAATCCCTGTGGGCCTTGC 60AATCCCTGTGGGCCTTGCTCAGA 61 AAGCATTTGTTTGTACAAGATCC 62AAGATCCGCAGACGTGTAAATGT 63 AAATGTTCCTGCAAAAACACAGA 64AATGTTCCTGCAAAAACACAGAC 65 AAAAACACAGACTCGCGTTGCAA 66AAAACACAGACTCGCGTTGCAAG 67 AAACACAGACTCGCGTTGCAAGG 68AACACAGACTCGCGTTGCAAGGC 69 AAGGCGAGGCAGCTTGAGTTAAA 70AAACGAACGTACTTGCAGATGTG 71 AACGAACGTACTTGCAGATGTGA 72AAGTGGTCCCAGGCTGCACCCAT 73 AAGGAGGAGGGCAGAATCATCAC 74AAGTGGTGAAGTTCATGGATGTC 75 AAAATCCCTGTGGGCCTTGCTCA 76

[0050] The siRNA of the invention can be obtained using a number oftechniques known to those of skill in the art. For example, the siRNAcan be chemically synthesized or recombinantly produced using methodsknown in the art, such as the Drosophila in vitro system described inU.S. published application 2002/0086356 of Tuschl et al., the entiredisclosure of which is herein incorporated by reference.

[0051] Preferably, the siRNA of the invention are chemically synthesizedusing appropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. The siRNA can be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

[0052] Alternatively, siRNA can also be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing siRNA of the invention from a plasmid include,for example, the U6 or H1 RNA pol III promoter sequences and thecytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant plasmids of the inventioncan also comprise inducible or regulatable promoters for expression ofthe siRNA in a particular tissue or in a particular intracellularenvironment.

[0053] The siRNA expressed from recombinant plasmids can either beisolated from cultured cell expression systems by standard techniques,or can be expressed intracellularly at or near the area ofneovascularization in vivo. The use of recombinant plasmids to deliversiRNA of the invention to cells in vivo is discussed in more detailbelow.

[0054] siRNA of the invention can be expressed from a recombinantplasmid either as two separate, complementary RNA molecules, or as asingle RNA molecule with two complementary regions.

[0055] Selection of plasmids suitable for expressing siRNA of theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the plasmid, and methods of delivering the recombinantplasmid to the cells of interest are within the skill in the art. See,for example Tuschl, T. (2002), Nat. Biotechnol, 20: 446-448; BrummelkampT R et al. (2002), Science 296: 550-553; Miyagishi M et al. (2002), Nat.Biotechnol. 20: 497-500; Paddison P J et al. (2002), Genes Dev. 16:948-958; Lee N S et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul CP et al. (2002), Nat. Biotechnol. 20: 505-508, the entire disclosures ofwhich are herein incorporated by reference.

[0056] A plasmid comprising nucleic acid sequences for expressing ansiRNA of the invention is described in Example 7 below. That plasmid,called pAAVsiRNA, comprises a sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and an antisense RNA strand coding sequencein operable connection with a polyT termination sequence under thecontrol of a human U6 RNA promoter. The plasmid pAAVsiRNA is ultimatelyintended for use in producing an recombinant adeno-associated viralvector comprising the same nucleic acid sequences for expressing ansiRNA of the invention.

[0057] As used herein, “in operable connection with a polyT terminationsequence” means that the nucleic acid sequences encoding the sense orantisense strands are immediately adjacent to the polyT terminationsignal in the 5′ direction. During transcription of the sense orantisense sequences from the plasmid, the polyT termination signals actto terminate transcription.

[0058] As used herein, “under the control” of a promoter means that thenucleic acid sequences encoding the sense or antisense strands arelocated 3′ of the promoter, so that the promoter can initiatetranscription of the sense or antisense coding sequences.

[0059] The siRNA of the invention can also be expressed from recombinantviral vectors intracellularly at or near the area of neovascularizationin vivo. The recombinant viral vectors of the invention comprisesequences encoding the siRNA of the invention and any suitable promoterfor expressing the siRNA sequences. Suitable promoters include, forexample, the U6 or H1 RNA pol III promoter sequences and thecytomegalovirus promoter. Selection of other suitable promoters iswithin the skill in the art. The recombinant viral vectors of theinvention can also comprise inducible or regulatable promoters forexpression of the siRNA in a particular tissue or in a particularintracellular environment. The use of recombinant viral vectors todeliver siRNA of the invention to cells in vivo is discussed in moredetail below.

[0060] siRNA of the invention can be expressed from a recombinant viralvector either as two separate, complementary RNA molecules, or as asingle RNA molecule with two complementary regions.

[0061] Any viral vector capable of accepting the coding sequences forthe siRNA molecule(s) to be expressed can be used, for example vectorsderived from adenovirus (AV); adeno-associated virus (AAV); retroviruses(e.g. lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpesvirus, and the like. The tropism of the viral vectors can also bemodified by pseudotyping the vectors with envelope proteins or othersurface antigens from other viruses. For example, an AAV vector of theinvention can be pseudotyped with surface proteins from vesicularstomatitis virus (VSV), rabies, Ebola, Mokola, and the like.

[0062] Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Domburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;and Anderson W F (1998), Nature 392: 25-30, the entire disclosures ofwhich are herein incorporated by reference.

[0063] Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the siRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orHI RNA promoters, or the cytomegalovirus (CMV) promoter.

[0064] A suitable AV vector for expressing the siRNA of the invention, amethod for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are described in Xia H et al.(2002), Nat. Biotech. 20: 1006-1010.

[0065] Suitable AAV vectors for expressing the siRNA of the invention,methods for constructing the recombinant AV vector, and methods fordelivering the vectors into target cells are described in Samulski R etal. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J.Virol., 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826;U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are herein incorporated byreference. An exemplary method for generating a recombinant AAV vectorof the invention is described in Example 7 below.

[0066] The ability of an siRNA containing a given target sequence tocause RNAi-mediated degradation of the target mRNA can be evaluatedusing standard techniques for measuring the levels of RNA or protein incells. For example, siRNA of the invention can be delivered to culturedcells, and the levels of target mRNA can be measured by Northern blot ordot blotting techniques, or by quantitative RT-PCR. Alternatively, thelevels of VEGF, Flt-1 or Flk-1/KDR receptor protein in the culturedcells can be measured by ELISA or Western blot. A suitable cell culturesystem for measuring the effect of the present siRNA on target mRNA orprotein levels is described in Example 1 below.

[0067] RNAi-mediated degradation of target mRNA by an siRNA containing agiven target sequence can also be evaluated with animal models ofneovascularization, such as the ROP or CNV mouse models. For example,areas of neovascularization in an ROP or CNV mouse can be measuredbefore and after administration of an siRNA. A reduction in the areas ofneovascularization in these models upon administration of the siRNAindicates the down-regulation of the target mRNA (see Example 6 below).

[0068] As discussed above, the siRNA of the invention target and causethe RNAi-mediated degradation of VEGF, Flt-1 or Flk-1/KDR mRNA, oralternative splice forms, mutants or cognates thereof. Degradation ofthe target mRNA by the present siRNA reduces the production of afunctional gene product from the VEGF, Flt-1 or Flk-1/KDR genes. Thus,the invention provides a method of inhibiting expression of VEGF, Flt-1or Flk-1/KDR in a subject, comprising administering an effective amountof an siRNA of the invention to the subject, such that the target mRNAis degraded. As the products of the VEGF, Flt-1 and Flk-1/KDR genes arerequired for initiating and maintaining angiogenesis, the invention alsoprovides a method of inhibiting angiogenesis in a subject by theRNAi-mediated degradation of the target mRNA by the present siRNA.

[0069] As used herein, a “subject” includes a human being or non-humananimal. Preferably, the subject is a human being.

[0070] As used herein, an “effective amount” of the siRNA is an amountsufficient to cause RNAi-mediated degradation of the target mRNA, or anamount sufficient to inhibit the progression of angiogenesis in asubject.

[0071] RNAi-mediated degradation of the target mRNA can be detected bymeasuring levels of the target mRNA or protein in the cells of asubject, using standard techniques for isolating and quantifying mRNA orprotein as described above.

[0072] Inhibition of angiogenesis can be evaluated by directly measuringthe progress of pathogenic or nonpathogenic angiogenesis in a subject;for example, by observing the size of a neovascularized area before andafter treatment with the siRNA of the invention. An inhibition ofangiogenesis is indicated if the size of the neovascularized area staysthe same or is reduced. Techniques for observing and measuring the sizeof neovascularized areas in a subject are within the skill in the art;for example, areas of choroid neovascularization can be observed byophthalmoscopy.

[0073] Inhibition of angiogenesis can also be inferred through observinga change or reversal in a pathogenic condition associated with theangiogenesis. For example, in ARMD, a slowing, halting or reversal ofvision loss indicates an inhibition of angiogenesis in the choroid. Fortumors, a slowing, halting or reversal of tumor growth, or a slowing orhalting of tumor metastasis, indicates an inhibition of angiogenesis ator near the tumor site. Inhibition of non-pathogenic angiogenesis canalso be inferred from, for example, fat loss or a reduction incholesterol levels upon administration of the siRNA of the invention.

[0074] It is understood that the siRNA of the invention can degrade thetarget mRNA (and thus inhibit angiogenesis) in substoichiometricamounts. Without wishing to be bound by any theory, it is believed thatthe siRNA of the invention causes degradation of the target mRNA in acatalytic manner. Thus, compared to standard anti-angiogenic therapies,significantly less siRNA needs to be delivered at or near the site ofneovascularization to have a therapeutic effect.

[0075] One skilled in the art can readily determine an effective amountof the siRNA of the invention to be administered to a given subject, bytaking into account factors such as the size and weight of the subject;the extent of the neovascularization or disease penetration; the age,health and sex of the subject; the route of administration; and whetherthe administration is regional or systemic. Generally, an effectiveamount of the siRNA of the invention comprises an intercellularconcentration at or near the neovascularization site of from about 1nanomolar (nM) to about 100 nM, preferably from about 2 nM to about 50nM, more preferably from about 2.5 nM to about 10 nM. It is contemplatedthat greater or lesser amounts of siRNA can be administered.

[0076] The present methods can be used to inhibit angiogenesis which isnon-pathogenic; i.e., angiogenesis which results from normal processesin the subject. Examples of non-pathogenic angiogenesis includeendometrial neovascularization, and processes involved in the productionof fatty tissues or cholesterol. Thus, the invention provides a methodfor inhibiting non-pathogenic angiogenesis, e.g., for controlling weightor promoting fat loss, for reducing cholesterol levels, or as anabortifacient.

[0077] The present methods can also inhibit angiogenesis which isassociated with an angiogenic disease; i.e., a disease in whichpathogenicity is associated with inappropriate or uncontrolledangiogenesis. For example, most cancerous solid tumors generate anadequate blood supply for themselves by inducing angiogenesis in andaround the tumor site. This tumor-induced angiogenesis is often requiredfor tumor growth, and also allows metastatic cells to enter thebloodstream.

[0078] Other angiogenic diseases include diabetic retinopathy,age-related macular degeneration (ARMD), psoriasis, rheumatoid arthritisand other inflammatory diseases. These diseases are characterized by thedestruction of normal tissue by newly formed blood vessels in the areaof neovascularization. For example, in ARMD, the choroid is invaded anddestroyed by capillaries. The angiogenesis-driven destruction of thechoroid in ARMD eventually leads to partial or full blindness.

[0079] Preferably, an siRNA of the invention is used to inhibit thegrowth or metastasis of solid tumors associated with cancers; forexample breast cancer, lung cancer, head and neck cancer, brain cancer,abdominal cancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma; skin cancer(e.g., melanoma), lymphomas and blood cancer.

[0080] More preferably, an siRNA of the invention is used to inhibitchoroidal neovascularization in age-related macular degeneration.

[0081] For treating angiogenic diseases, the siRNA of the invention canadministered to a subject in combination with a pharmaceutical agentwhich is different from the present siRNA. Alternatively, the siRNA ofthe invention can be administered to a subject in combination withanother therapeutic method designed to treat the angiogenic disease. Forexample, the siRNA of the invention can be administered in combinationwith therapeutic methods currently employed for treating cancer orpreventing tumor metastasis (e.g., radiation therapy, chemotherapy, andsurgery). For treating tumors, the siRNA of the invention is preferablyadministered to a subject in combination with radiation therapy, or incombination with chemotherapeutic agents such as cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

[0082] In the present methods, the present siRNA can be administered tothe subject either as naked siRNA, in conjunction with a deliveryreagent, or as a recombinant plasmid or viral vector which expresses thesiRNA.

[0083] Suitable delivery reagents for administration in conjunction withthe present siRNA include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes. A preferred delivery reagent is a liposome.

[0084] Liposomes can aid in the delivery of the siRNA to a particulartissue, such as retinal or tumor tissue, and can also increase the bloodhalf-life of the siRNA. Liposomes suitable for use in the invention areformed from standard vesicle-forming lipids, which generally includeneutral or negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. A variety of methods are known forpreparing liposomes, for example as described in Szoka et al. (1980),Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which areherein incorporated by reference.

[0085] Preferably, the liposomes encapsulating the present siRNAcomprises a ligand molecule that can target the liposome to a particularcell or tissue at or near the site of angiogenesis. Ligands which bindto receptors prevalent in tumor or vascular endothelial cells, such asmonoclonal antibodies that bind to tumor antigens or endothelial cellsurface antigens, are preferred.

[0086] Particularly preferably, the liposomes encapsulating the presentsiRNA are modified so as to avoid clearance by the mononuclearmacrophage and reticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

[0087] Opsonization-inhibiting moieties for use in preparing theliposomes of the invention are typically large hydrophilic polymers thatare bound to the liposome membrane. As used herein, an opsonizationinhibiting moiety is “bound” to a liposome membrane when it ischemically or physically attached to the membrane, e.g., by theintercalation of a lipid-soluble anchor into the membrane itself, or bybinding directly to active groups of membrane lipids. Theseopsonization-inhibiting hydrophilic polymers form a protective surfacelayer which significantly decreases the uptake of the liposomes by themacrophage-monocyte system (“MMS”) and reticuloendothelial system(“RES”); e.g., as described in U.S. Pat. No. 4,920,016, the entiredisclosure of which is herein incorporated by reference. Liposomesmodified with opsonization-inhibition moieties thus remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes.

[0088] Stealth liposomes are known to accumulate in tissues fed byporous or “leaky” microvasculature. Thus, target tissue characterized bysuch microvasculature defects, for example solid tumors, willefficiently accumulate these liposomes; see Gabizon, et al. (1988),P.N.A.S., USA, 18: 6949-53. In addition, the reduced uptake by the RESlowers the toxicity of stealth liposomes by preventing significantaccumulation in the liver and spleen. Thus, liposomes of the inventionthat are modified with opsonization-inhibition moieties can deliver thepresent siRNA to tumor cells.

[0089] Opsonization inhibiting moieties suitable for modifying liposomesare preferably water-soluble polymers with a molecular weight from about500 to about 40,000 daltons, and more preferably from about 2,000 toabout 20,000 daltons. Such polymers include polyethylene glycol (PEG) orpolypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, andPEG or PPG stearate; synthetic polymers such as polyacrylamide or polyN-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines;polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitolto which carboxylic or amino groups are chemically linked, as well asgangliosides, such as ganglioside GM₁. Copolymers of PEG, methoxy PEG,or methoxy PPG, or derivatives thereof, are also suitable. In addition,the opsonization inhibiting polymer can be a block copolymer of PEG andeither a polyamino acid, polysaccharide, polyamidoamine,polyethyleneamine, or polynucleotide. The opsonization inhibitingpolymers can also be natural polysaccharides containing amino acids orcarboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronicacid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid,carrageenan; laminated polysaccharides or oligosaccharides (linear orbranched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

[0090] Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

[0091] The opsonization inhibiting moiety can be bound to the liposomemembrane by any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

[0092] Recombinant plasmids which express siRNA of the invention arediscussed above. Such recombinant plasmids can also be administereddirectly or in conjunction with a suitable delivery reagent, includingthe Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine;cellfectin; polycations (e.g., polylysine) or liposomes. Recombinantviral vectors which express siRNA of the invention are also discussedabove, and methods for delivering such vectors to an area ofneovascularization in a patient are within the skill in the art.

[0093] The siRNA of the invention can be administered to the subject byany means suitable for delivering the siRNA to the cells of the tissueat or near the area of neovascularization. For example, the siRNA can beadministered by gene gun, electroporation, or by other suitableparenteral or enteral administration routes.

[0094] Suitable enteral administration routes include oral, rectal, orintranasal delivery.

[0095] Suitable parenteral administration routes include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition including subcutaneous infusion (such as by osmotic pumps);direct application to the area at or near the site ofneovascularization, for example by a catheter or other placement device(e.g., a retinal pellet or a suppository or an implant comprising aporous, non-porous, or gelatinous material); and inhalation. It ispreferred that injections or infusions of the siRNA be given at or nearthe site of neovascularization.

[0096] The siRNA of the invention can be administered in a single doseor in multiple doses. Where the administration of the siRNA of theinvention is by infusion, the infusion can be a single sustained dose orcan be delivered by multiple infusions. Injection of the agent directlyinto the tissue is at or near the site of neovascularization preferred.Multiple injections of the agent into the tissue at or near the site ofneovascularization are particularly preferred.

[0097] One skilled in the art can also readily determine an appropriatedosage regimen for administering the siRNA of the invention to a givensubject. For example, the siRNA can be administered to the subject once,for example as a single injection or deposition at or near theneovascularization site. Alternatively, the siRNA can be administeredonce or twice daily to a subject for a period of from about three toabout twenty-eight days, more preferably from about seven to about tendays. In a preferred dosage regimen, the siRNA is injected at or nearthe site of neovascularization once a day for seven days. Where a dosageregimen comprises multiple administrations, it is understood that theeffective amount of siRNA administered to the subject can comprise thetotal amount of siRNA administered over the entire dosage regimen.

[0098] The siRNA of the invention are preferably formulated aspharmaceutical compositions prior to administering to a subject,according to techniques known in the art. Pharmaceutical compositions ofthe present invention are characterized as being at least sterile andpyrogen-free. As used herein, “pharmaceutical formulations” includeformulations for human and veterinary use. Methods for preparingpharmaceutical compositions of the invention are within the skill in theart, for example as described in Remington's Pharmaceutical Science,17th ed., Mack Publishing Company, Easton, Pa. (1985), the entiredisclosure of which is herein incorporated by reference.

[0099] The present pharmaceutical formulations comprise an siRNA of theinvention (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and thelike.

[0100] Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

[0101] For solid compositions, conventional nontoxic solid carriers canbe used; for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like.

[0102] For example, a solid pharmaceutical composition for oraladministration can comprise any of the carriers and excipients listedabove and 10-95%, preferably 25%-75%, of one or more siRNA of theinvention. A pharmaceutical composition for aerosol (inhalational)administration can comprise 0.01-20% by weight, preferably 1%-10% byweight, of one or more siRNA of the invention encapsulated in a liposomeas described above, and propellant. A carrier can also be included asdesired; e.g., lecithin for intranasal delivery.

[0103] The invention will now be illustrated with the followingnon-limiting examples. All animal experiments discussed below wereperformed using the University of Pennsylvania institutional guidelinesfor the care and use of animals in research.

EXAMPLE 1 siRNA Transfection and Hypoxia Induction In Vitro

[0104] siRNA Design—A 19 nt sequence located 329 nt from the 5′ end ofhuman VEGF mRNA was chosen as a target sequence: AAACCTCACCAAGGCCAGCAC(SEQ ID NO: 51). To ensure that it was not contained in the mRNA fromany other genes, this target sequence was entered into the BLAST searchengine provided by NCBI. The use of the BLAST algorithm is described inAltschul et al. (1990), J. Mol. Biol. 215: 403-410 and Altschul et al.(1997), Nucleic Acids Res. 25: 3389-3402, the disclosures of which areherein incorporated by reference in their entirety. As no other mRNA wasfound which contained the target sequence, an siRNA duplex wassynthesized to target this sequence (Dharmacon Research, Inc.,Lafayette, Colo.).

[0105] The siRNA duplex had the following sense and antisense strands.

[0106] Sense:

[0107] 5′-accucaccaaggccagcacTT-3′ (SEQ ID NO: 77).

[0108] Antisense:

[0109] 5′-gugcuggccuuggugagguTT-3′ (SEQ ID NO: 78).

[0110] Together, the siRNA sense and antisense strands formed a 19 ntdouble-stranded siRNA with TT 3′ overhangs (shown in bold) on eachstrand. This siRNA was termed “Candidate 5.” Other siRNA which targethuman VEGF mRNA were designed and tested as described for Candidate 5.

[0111] An siRNA targeting the following sequence in green fluorescentprotein (GFP) mRNA was used as a nonspecific control: GGCTACGTCCAGCGCACC(SEQ ID NO: 79). The siRNA was purchased from Dharmacon (Lafayette,Colo.).

[0112] siRNA Transfection and Hypoxia Induction In Vitro—Human celllines (293; Hela and ARPE19) were separately seeded into 24-well platesin 250 microliters of complete DMEM medium one day prior totransfection, so that the cells were ˜50% confluent at the time oftransfection. Cells were transfected with 2.5 nM Candidate 5 siRNA, andwith either no siRNA or 2.5 nM non-specific siRNA (targeting GFP) ascontrols. Transfections were performed in all cell lines with the“Transit TKO Transfection” reagent, as recommended by the manufacturer(Mirus).

[0113] Twenty four hours after transfection, hypoxia was induced in thecells by the addition of desferoxamide mesylate to a final concentrationof 130 micromolar in each well. Twenty four hours post-transfection, thecell culture medium was removed from all wells, and a human VEGF ELISA(R&D systems, Minneapolis, Minn.) was performed on the culture medium asdescribed in the Quantikine human VEGF ELISA protocol available from themanufacturer, the entire disclosure of which is herein incorporated byreference.

[0114] As can be seen in FIG. 1, RNAi degradation induced by Candidate 5siRNA significantly reduces the concentration of VEGF produced by thehypoxic 293 and HeLa cells. There was essentially no difference in theamount of VEGF produced by hypoxic cells treated with either no siRNA orthe non-specific siRNA control. Similar results were also seen withhuman ARPE19 cells treated under the same conditions. Thus, RNAinterference with VEGF-targeted siRNA disrupts the pathogenicup-regulation of VEGF in human cultured cells in vitro.

[0115] The experiment outlined above was repeated on mouse NIH 3T3 cellsusing a mouse-specific VEGF siRNA (see Example 6 below), and VEGFproduction was quantified with a mouse VEGF ELISA (R&D systems,Minneapolis, Minn.) as described in the Quantikine mouse VEGF ELISAprotocol available from the manufacturer, the entire disclosure of whichis herein incorporated by reference. Results similar to those reportedin FIG. 1 for the human cell lines were obtained.

EXAMPLE 2 Effect of Increasing siRNA Concentration on VEGF Production inHuman Cultured Cells

[0116] The experiment outlined in Example 1 was repeated with human 293,HeLa and ARPE19 cells using a range of siRNA concentrations from 10 nMto 50 nM. The ability of the Candidate 5 siRNA to down-regulate VEGFproduction increased moderately up to approximately 13 nM siRNA, but aplateau effect was seen above this concentration. These resultshighlight the catalytic nature of siRNA-mediated RNAi degradation ofmRNA, as the plateau effect appears to reflect VEGF production from thefew cells not transfected with the siRNA. For the majority of cellswhich had been transfected with the siRNA, the increased VEGF mRNAproduction induced by the hypoxia is outstripped by the siRNA-induceddegradation of the target mRNA at siRNA concentrations greater thanabout 13 nM.

EXAMPLE 3 Specificity of siRNA Targeting

[0117] NIH 3T3 mouse fibroblasts were grown in 24-well plates understandard conditions, so that the cells were ˜50% confluent one day priorto transfection. The human VEGF siRNA Candidate 5 was transfected into aNIH 3T3 mouse fibroblasts as in Example 1. Hypoxia was then induced inthe transfected cells, and murine VEGF concentrations were measured byELISA as in Example 1.

[0118] The sequence targeted by the human VEGF siRNA Candidate 5 differsfrom the murine VEGF mRNA by one nucleotide. As can be seen in FIG. 2,the human VEGF siRNA has no affect on the ability of the mouse cells toup-regulate mouse VEGF after hypoxia. These results show that siRNAinduced RNAi degradation is sequence-specific to within a one nucleotideresolution.

EXAMPLE 4 In Vivo Delivery of siRNA to Murine Retinal Pigment EpithelialCells

[0119] VEGF is upregulated in the retinal pigment epithelial (RPE) cellsof human patients with age-related macular degeneration (ARMD). To showthat functional siRNA can be delivered to RPE cells in vivo, weexpressed GFP in mouse retinas with a recombinant adenovirus, andsilenced GFP expression with siRNA. The experiment was conducted asfollows.

[0120] One eye from each of five adult C57/Black6 mice (Jackson Labs,Bar Harbor, Me.) was injected subretinally as described in Bennett etal. (1996), Hum. Gene Ther. 7: 1763-1769, the entire disclosure of whichis herein incorporated by reference, with a mixture containing ˜1×10⁸particles of adenovirus containing eGFP driven by the CMV promoter and20 picomoles of siRNA targeting eGFP conjugated with transit TKO reagent(Mirus).

[0121] As positive control, the contralateral eyes were injected with amixture containing ˜1×10⁸ particles of adenovirus containing eGFP drivenby the CMV promoter and 20 picomoles of siRNA targeting human VEGFconjugated with transit TKO reagent (Mirus). Expression of GFP wasdetected by fundus ophthalmoscopy 48 hours and 60 hours after injection.Animals were sacrificed at either 48 hours or 60 hours post-injection.The eyes were enucleated and fixed in 4% paraformaldehyde, and wereprepared either as flat mounts or were processed into 10 microncryosections for fluorescent microscopy.

[0122] No GFP fluorescence was detectable by ophthalmoscopy in the eyeswhich received the siRNA targeted to GFP mRNA in 4 out of 5 mice,whereas GFP fluorescence was detectable in the contralateral eye whichreceived the non-specific control siRNA. A representative flat mountanalyzed by fluorescence microscopy showed a lack of GFP fluorescence inthe eye which received GFP siRNA, as compared to an eye that receivedthe non-specific control siRNA. Cryosections of another retina showedthat the recombinant adenovirus efficiently targets the RPE cells, andwhen the adenovirus is accompanied by siRNA targeted to GFP mRNA,expression of the GFP transgene is halted.

[0123] While there is some GFP fluorescence detectable by fluorescencemicroscopy in eyes that received siRNA targeted to GFP mRNA, thefluorescence is greatly suppressed as compared to controls that receivednon-specific siRNA. These data demonstrate that functional siRNA can bedelivered in vivo to RPE cells.

EXAMPLE 5 In Vivo Expression and siRNA-Induced RNAi Degradation of HumanVEGF in Murine Retinas

[0124] In order to demonstrate that siRNA targeted to VEGF functioned invivo, an exogenous human VEGF expression cassette was delivered to mouseRPE cells via an adenovirus by subretinal injection, as in Example 4.One eye received Candidate 5 siRNA, and the contralateral eye receivedsiRNA targeted to GFP mRNA. The animals were sacrificed 60 hourspost-injection, and the injected eyes were removed and snap frozen inliquid N₂ following enucleation. The eyes were then homogenized in lysisbuffer, and total protein was measured using a standard Bradford proteinassay (Roche, Germany). The samples were normalized for total proteinprior to assaying for human VEGF by ELISA as described in Example 1.

[0125] The expression of VEGF was somewhat variable from animal toanimal. The variability of VEGF levels correlated well to those observedin the GFP experiments of Example 4, and can be attributed to some errorfrom injection to injection, and the differential ability of adenovirusto delivery the target gene in each animal. However, there was asignificant attenuation of VEGF expression in each eye that receivedVEGF siRNA, as compared to the eyes receiving the non-specific controlsiRNA (FIG. 4). These data indicate that the Candidate 5 siRNA waspotent and effective in silencing human VEGF expression in murine RPEcells in vivo.

EXAMPLE 6 Inhibition of Choroidal Neovascularization in the Mouse CNVModel

[0126] There is evidence that choroidal neovascularization in ARMD isdue to the upregulation of VEGF in the RPE cells. This human pathologiccondition can be modeled in the mouse by using a laser to burn a spot onthe retina. During the healing process, VEGF is believed to beup-regulated in the RPE cells of the burned region, leading tore-vascularization of the choroid. This model is called the mouse CNV(“choroidal neovascularization”) model.

[0127] For rescue of the mouse CNV model, a mouse siRNA was designedthat incorporated a one nucleotide change from the human “Candidate 5”siRNA from Example 1. The mouse siRNA specifically targeted mouse VEGFmRNA at the sequence AAACCUCACCAAAGCCAGCAC (SEQ ID NO: 80). Other siRNAthat target mouse VEGF were also designed and tested. The GFP siRNA usedas a nonspecific control in Example 1 was also used as a non-specificcontrol here.

[0128] Twenty four hours after laser treatment, one eye from each ofeleven adult C57/Black6 mice (Jackson Labs, Bar Harbor, Me.) wasinjected subretinally with a mixture containing ˜1×10⁸ particles ofadenovirus containing LacZ driven by the CMV promoter and 20 picomolesof siRNA targeting mouse VEGF conjugated with transit TKO reagent(Mirus), as in Example 4. As a control, contralateral eyes received amixture containing ˜1×10⁸ particles of adenovirus containing LacZ drivenby the CMV promoter and 20 picomoles of siRNA targeting GFP conjugatedwith transit TKO reagent (Mirus).

[0129] Fourteen days after the laser treatment, the mice were perfusedwith fluorescein and the area of neovascularization was measured aroundthe burn spots. Areas of the burn spots in the contra-lateral eye wereused as a control. The site of neovascularization around the burn spotsin animals that received siRNA targeting mouse VEGF was, on average, ¼the area of the control areas. These data support the use of VEGFdirected siRNA for therapy of ARMD.

EXAMPLE 7 Generation of an Adeno-Associated Viral Vector for Expressionof siRNA

[0130] A “cis-acting” plasmid for generating a recombinant AAV vectorfor delivering an siRNA of the invention was generated by PCR basedsubcloning, essentially as described in Samulski R et al. (1987), supra.The cis-acting plasmid was called “pAAVsiRNA.”

[0131] The rep and cap genes of psub201 were replaced with the followingsequences in this order: a 19 nt sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and a 19 nt antisense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter. A schematic representation ofpAAVsiRNA is given if FIG. 5.

[0132] A recombinant AAV siRNA vector was obtained by transfectingpAAVsiRNA into human 293 cells previously infected with E1-deletedadenovirus, as described in Fisher K J et al. (1996), supra. The AAV repand cap functions were provided by a trans-acting plasmid pAAV/Ad asdescribed in Samulski R et al. (1989), supra. Production lots of therecombinant AAV siRNA vector were titered according to the number ofgenome copies/ml, as described in Fisher K J et al. (1996), supra.

1 80 1 2250 DNA Mus musculus 1 tgagccaggc tggcaggaag gagcctccctcagggtttcg ggaaccagac ctctcaccgg 60 aaagaccgat taaccatgtc accaccacgccatcatcgtc accgttgaca gaacagtcct 120 taatccagaa agcctgacat gaaggaagaggagactcttc gaggagcact ttgggtccgg 180 agggcgagac tccggcagac gcattcccgggcaggtgacc aagcacggtc cctcgtggga 240 ctggattcgc cattttctta tatctgctgctaaatcgcca agcccggaag attagggttg 300 tttctgggat tcctgtagac acacccacccacatacacac atatatatat attatatata 360 taaataaata tatatgtttt atatataaaatatatatata ttcttttttt taaattaact 420 ctgctaatgt tattggtgtc ttcactggatatgtttgact gctgtggact tgtgttggga 480 ggaggatgtc ctcactcgga tgccgacatgggagacaatg ggatgaaagg cttcagtgtg 540 gtctgagaga ggccgaagtc cttttgcctgccggggagca agcaaggcca gggcacgggg 600 gcacattggc tcacttccag aaacacgacaaacccattcc tggccctgag tcaagaggac 660 agagagacag atgatgacac agaaagagataaagatgccg gttccaacca gaagtttggg 720 gagcctcagg acatggcatg ctttgtggatccccatgata gtctacaaaa gcaccccgcc 780 cctctgggca ctgcctggaa gaatcgggagcctggccagc cttcagctcg ctcctccact 840 tctgaggggc ctaggaggcc tcccacaggtgtcccggcaa gagaagacac ggtggtggaa 900 gaagaggcct ggtaatggcc cctcctcctgggaccccttc gtcctctcct taccccacct 960 cctgggtaca gcccaggagg accttgtgtgatcagaccat tgaaaccact aattctgtcc 1020 ccaggagact tggctctgtg tgtgagtggcttacccttcc tcatcttccc ttcccaaggc 1080 acagagcaat ggggcaggac ccgcaagcccctcacggagg cagagaaaag agaaagtgtt 1140 ttatatacgg tacttattta atagccctttttaattagaa attaaaacag ttaatttaat 1200 taaagagtag ggtttttttc agtattcttggttaatattt aatttcaact atttatgaga 1260 tgtatctctc gctctctctt atttgtacttatgtgtgtgt gtgtgtgtgt gtgtgtgtgt 1320 gtgtgtgtgt gtatgaaatc tgtgtttccaatctctctct cccagatcgg tgacagtcac 1380 tagcttgtcc tgagaagata tttaattttgctaacactca gctctgccct cccttgtccc 1440 caccacacat tcctttgaaa taaggtttcaatatacattt acatactata tatatatttg 1500 gcaacttgtg tttgtatata aatatatatatatatatatg tttatgtata tatgtgattc 1560 tgataaaata gacattgcta ttctgttttttatatgtaaa aacaaaacaa gaaaaataga 1620 gaattctaca tactaaatct ctctccttttttaattttaa tatttgttat catttattta 1680 ttggtgctac tgtttatccg taataattgtgggggaaaaa gatattaaca tcacgtcttt 1740 gtctctagag cagttttccg agatattccgtagtacatat ttatttttaa acagcaacaa 1800 agaaatacag atatatctta aaaaaaaagcattttgtatt aaagaattga attctgatct 1860 caaagctctc cctggtctct ccttctctcctgggccctcc tgtctcgctt tccctcctcc 1920 tttggggtac atagtttttg tcttaggtttgagaagcagt ccctggagta gaatatgggg 1980 tgacccatcc attcctgggc ggaggggagatggctccttt gccaagggtc ctcacactac 2040 gtggtactct gttccttgtc agacaaggatgggggcatgt ctccaggtgc taactggaga 2100 tcggagagag ctgttggctg cagctggccaggatttgggc atgccgggga catgggaggc 2160 tgtgagccca gcatgcagtt tacttctgggtgctaaatgg aagagtccag taaaaagagt 2220 cttgcccatg ggattccatt ccgctttgtg2250 2 444 DNA Homo sapiens 2 atgaactttc tgctgtcttg ggtgcattggagccttgcct tgctgctcta cctccaccat 60 gccaagtggt cccaggctgc acccatggcagaaggaggag ggcagaatca tcacgaagtg 120 gtgaagttca tggatgtcta tcagcgcagctactgccatc caatcgagac cctggtggac 180 atcttccagg agtaccctga tgagatcgagtacatcttca agccatcctg tgtgcccctg 240 atgcgatgcg ggggctgctg caatgacgagggcctggagt gtgtgcccac tgaggagtcc 300 aacatcacca tgcagattat gcggatcaaacctcaccaag gccagcacat aggagagatg 360 agcttcctac agcacaacaa atgtgaatgcagaccaaaga aagatagagc aagacaagaa 420 aaatgtgaca agccgaggcg gtga 444 3576 DNA Homo sapiens 3 atgaactttc tgctgtcttg ggtgcattgg agccttgccttgctgctcta cctccaccat 60 gccaagtggt cccaggctgc acccatggca gaaggaggagggcagaatca tcacgaagtg 120 gtgaagttca tggatgtcta tcagcgcagc tactgccatccaatcgagac cctggtggac 180 atcttccagg agtaccctga tgagatcgag tacatcttcaagccatcctg tgtgcccctg 240 atgcgatgcg ggggctgctg caatgacgag ggcctggagtgtgtgcccac tgaggagtcc 300 aacatcacca tgcagattat gcggatcaaa cctcaccaaggccagcacat aggagagatg 360 agcttcctac agcacaacaa atgtgaatgc agaccaaagaaagatagagc aagacaagaa 420 aatccctgtg ggccttgctc agagcggaga aagcatttgtttgtacaaga tccgcagacg 480 tgtaaatgtt cctgcaaaaa cacagactcg cgttgcaaggcgaggcagct tgagttaaac 540 gaacgtactt gcagatgtga caagccgagg cggtga 576 4648 DNA Homo sapiens 4 atgaactttc tgctgtcttg ggtgcattgg agccttgccttgctgctcta cctccaccat 60 gccaagtggt cccaggctgc acccatggca gaaggaggagggcagaatca tcacgaagtg 120 gtgaagttca tggatgtcta tcagcgcagc tactgccatccaatcgagac cctggtggac 180 atcttccagg agtaccctga tgagatcgag tacatcttcaagccatcctg tgtgcccctg 240 atgcgatgcg ggggctgctg caatgacgag ggcctggagtgtgtgcccac tgaggagtcc 300 aacatcacca tgcagattat gcggatcaaa cctcaccaaggccagcacat aggagagatg 360 agcttcctac agcacaacaa atgtgaatgc agaccaaagaaagatagagc aagacaagaa 420 aaaaaatcag ttcgaggaaa gggaaagggg caaaaacgaaagcgcaagaa atcccggtat 480 aagtcctgga gcgttccctg tgggccttgc tcagagcggagaaagcattt gtttgtacaa 540 gatccgcaga cgtgtaaatg ttcctgcaaa aacacagactcgcgttgcaa ggcgaggcag 600 cttgagttaa acgaacgtac ttgcagatgt gacaagccgaggcggtga 648 5 670 DNA Homo sapiens 5 gccttgctgc tctacctcca ccatgccaagtggtcccagg ctgcacccat ggcagaagga 60 ggagggcaga atcatcacga agtggtgaagttcatggatg tctatcagcg cagctactgc 120 catccaatcg agaccctggt ggacatcttccaggagtacc ctgatgagat cgagtacatc 180 ttcaagccat cctgtgtgcc cctgatgcgatgcgggggct gctgcaatga cgagggcctg 240 gagtgtgtgc ccactgagga gtccaacatcaccatgcaga ttatgcggat caaacctcac 300 caaggccagc acataggaga gatgagcttcctacagcaca acaaatgtga atgcagacca 360 aagaaggata gagcaagaca agaaaaaaaatcagttcgag gaaagggaaa ggggcaaaaa 420 cgaaagcgca agaaatcccg gtataagtcctggagcgttt acgttggtgc ccgctgctgt 480 ctaatgccct ggagcctccc tggcccccatccctgtgggc cttgctcaga gcggagaaag 540 catttgtttg tacaagatcc gcagacgtgtaaatgttcct gcaaaaacac agactcgcgt 600 tgcaaggcga ggcagcttga gttaaacgaacgtacttgca gatgtgacaa gccgaggcgg 660 tgatgaatga 670 6 1137 DNA Homosapiens 6 atgctcattg tccagactgg ggtcagatca gcaaacaaag ggcctctgatggtgattgtt 60 gaatattgca aatatggaaa tctatccaac tacctcaaga gcaaatatgacttatttttt 120 ctcgacaagg atgtggcatc acacatggag cgtaagaaag aaaaaatggagccaggcctg 180 gaacaaggca agaaaccaaa actagatagc atcaccagca gcgagagctttgggagctcc 240 aagtttcagg aagataaaaa tctgagtgat gttgaggaag aggaggattctgatggtttc 300 taccaggagc ccatcactat ggaagatctg atttcttaca gttttcaagtggccagaggc 360 atgaagtttc tgtcttccag aaagtgcatt cattgggacc tggcagcaagaaacattctt 420 ttatctgaga acaatgtggt gaagatttgt gattttggcc ttgcccaggatatttacaag 480 aacgccgatt atgtgagaaa aggaggtggg tctccatacc caggagtgcaaatggatgag 540 cacttctgca gttgcctgag ggaaggcatg aggatgagag ctgctgagtactccactcct 600 gaaatctatc agatcatgct ggactgcagg cacaaagacc caaaagaaaggccaagattt 660 gcagaacttg tggaaaaact agaaaatagt gggtttacat actcaactcctgccttctct 720 gaggacttct tcaaggaagg tatttcagct cccaagttta gttcaggaagctctgatgat 780 gtcagatacg taaatgcttt caagttcatg agcctggaaa gaatcaaaacctttgaagaa 840 cttttgccaa atgccacctc catgtttgat gactaccagg gggacagcagcgctctgctg 900 gcctctccca tgctgaagcg cttcaccagg actgacagca aacccaaggcctcgctcaag 960 attgacttga gactaactag caaaagtaag aagtcggggc tttctgatgtcagcaggccc 1020 agtttctgcc attccaacag tgggcacatc agcaaaggca agggcaggttcacctacgac 1080 aacgccgagc tggaaaggaa gacggcgtgc tgctccccgc ccctctgggagttgtag 1137 7 5830 DNA Homo sapiens 7 actgagtccc gggaccccgg gagagcggtcagtgtgtggt cgctgcgttt cctctgcctg 60 cgccgggcat cacttgcgcg ccgcagaaagtccgtctggc agcctggata tcctctccta 120 ccggcacccg cagacgcccc tgcagccgccggtcggcgcc cgggctccct agccctgtgc 180 gctcaactgt cctgcgctgc ggggtgccgcgagttccacc tccgcgcctc cttctctaga 240 caggcgctgg gagaaagaac cggctcccgagttctgggca tttcgcccgg ctcgaggtgc 300 aggatgcaga gcaaggtgct gctggccgtcgccctgtggc tctgcgtgga gacccgggcc 360 gcctctgtgg gtttgcctag tgtttctcttgatctgccca ggctcagcat acaaaaagac 420 atacttacaa ttaaggctaa tacaactcttcaaattactt gcaggggaca gagggacttg 480 gactggcttt ggcccaataa tcagagtggcagtgagcaaa gggtggaggt gactgagtgc 540 agcgatggcc tcttctgtaa gacactcacaattccaaaag tgatcggaaa tgacactgga 600 gcctacaagt gcttctaccg ggaaactgacttggcctcgg tcatttatgt ctatgttcaa 660 gattacagat ctccatttat tgcttctgttagtgaccaac atggagtcgt gtacattact 720 gagaacaaaa acaaaactgt ggtgattccatgtctcgggt ccatttcaaa tctcaacgtg 780 tcactttgtg caagataccc agaaaagagatttgttcctg atggtaacag aatttcctgg 840 gacagcaaga agggctttac tattcccagctacatgatca gctatgctgg catggtcttc 900 tgtgaagcaa aaattaatga tgaaagttaccagtctatta tgtacatagt tgtcgttgta 960 gggtatagga tttatgatgt ggttctgagtccgtctcatg gaattgaact atctgttgga 1020 gaaaagcttg tcttaaattg tacagcaagaactgaactaa atgtggggat tgacttcaac 1080 tgggaatacc cttcttcgaa gcatcagcataagaaacttg taaaccgaga cctaaaaacc 1140 cagtctggga gtgagatgaa gaaatttttgagcaccttaa ctatagatgg tgtaacccgg 1200 agtgaccaag gattgtacac ctgtgcagcatccagtgggc tgatgaccaa gaagaacagc 1260 acatttgtca gggtccatga aaaaccttttgttgcttttg gaagtggcat ggaatctctg 1320 gtggaagcca cggtggggga gcgtgtcagaatccctgcga agtaccttgg ttacccaccc 1380 ccagaaataa aatggtataa aaatggaataccccttgagt ccaatcacac aattaaagcg 1440 gggcatgtac tgacgattat ggaagtgagtgaaagagaca caggaaatta cactgtcatc 1500 cttaccaatc ccatttcaaa ggagaagcagagccatgtgg tctctctggt tgtgtatgtc 1560 ccaccccaga ttggtgagaa atctctaatctctcctgtgg attcctacca gtacggcacc 1620 actcaaacgc tgacatgtac ggtctatgccattcctcccc cgcatcacat ccactggtat 1680 tggcagttgg aggaagagtg cgccaacgagcccagccaag ctgtctcagt gacaaaccca 1740 tacccttgtg aagaatggag aagtgtggaggacttccagg gaggaaataa aattgaagtt 1800 aataaaaatc aatttgctct aattgaaggaaaaaacaaaa ctgtaagtac ccttgttatc 1860 caagcggcaa atgtgtcagc tttgtacaaatgtgaagcgg tcaacaaagt cgggagagga 1920 gagagggtga tctccttcca cgtgaccaggggtcctgaaa ttactttgca acctgacatg 1980 cagcccactg agcaggagag cgtgtctttgtggtgcactg cagacagatc tacgtttgag 2040 aacctcacat ggtacaagct tggcccacagcctctgccaa tccatgtggg agagttgccc 2100 acacctgttt gcaagaactt ggatactctttggaaattga atgccaccat gttctctaat 2160 agcacaaatg acattttgat catggagcttaagaatgcat ccttgcagga ccaaggagac 2220 tatgtctgcc ttgctcaaga caggaagaccaagaaaagac attgcgtggt caggcagctc 2280 acagtcctag agcgtgtggc acccacgatcacaggaaacc tggagaatca gacgacaagt 2340 attggggaaa gcatcgaagt ctcatgcacggcatctggga atccccctcc acagatcatg 2400 tggtttaaag ataatgagac ccttgtagaagactcaggca ttgtattgaa ggatgggaac 2460 cggaacctca ctatccgcag agtgaggaaggaggacgaag gcctctacac ctgccaggca 2520 tgcagtgttc ttggctgtgc aaaagtggaggcatttttca taatagaagg tgcccaggaa 2580 aagacgaact tggaaatcat tattctagtaggcacggcgg tgattgccat gttcttctgg 2640 ctacttcttg tcatcatcct acggaccgttaagcgggcca atggagggga actgaagaca 2700 ggctacttgt ccatcgtcat ggatccagatgaactcccat tggatgaaca ttgtgaacga 2760 ctgccttatg atgccagcaa atgggaattccccagagacc ggctgaagct aggtaagcct 2820 cttggccgtg gtgcctttgg ccaagtgattgaagcagatg cctttggaat tgacaagaca 2880 gcaacttgca ggacagtagc agtcaaaatgttgaaagaag gagcaacaca cagtgagcat 2940 cgagctctca tgtctgaact caagatcctcattcatattg gtcaccatct caatgtggtc 3000 aaccttctag gtgcctgtac caagccaggagggccactca tggtgattgt ggaattctgc 3060 aaatttggaa acctgtccac ttacctgaggagcaagagaa atgaatttgt cccctacaag 3120 accaaagggg cacgattccg tcaagggaaagactacgttg gagcaatccc tgtggatctg 3180 aaacggcgct tggacagcat caccagtagccagagctcag ccagctctgg atttgtggag 3240 gagaagtccc tcagtgatgt agaagaagaggaagctcctg aagatctgta taaggacttc 3300 ctgaccttgg agcatctcat ctgttacagcttccaagtgg ctaagggcat ggagttcttg 3360 gcatcgcgaa agtgtatcca cagggacctggcggcacgaa atatcctctt atcggagaag 3420 aacgtggtta aaatctgtga ctttggcttggcccgggata tttataaaga tccagattat 3480 gtcagaaaag gagatgctcg cctccctttgaaatggatgg ccccagaaac aatttttgac 3540 agagtgtaca caatccagag tgacgtctggtcttttggtg ttttgctgtg ggaaatattt 3600 tccttaggtg cttctccata tcctggggtaaagattgatg aagaattttg taggcgattg 3660 aaagaaggaa ctagaatgag ggcccctgattatactacac cagaaatgta ccagaccatg 3720 ctggactgct ggcacgggga gcccagtcagagacccacgt tttcagagtt ggtggaacat 3780 ttgggaaatc tcttgcaagc taatgctcagcaggatggca aagactacat tgttcttccg 3840 atatcagaga ctttgagcat ggaagaggattctggactct ctctgcctac ctcacctgtt 3900 tcctgtatgg aggaggagga agtatgtgaccccaaattcc attatgacaa cacagcagga 3960 atcagtcagt atctgcagaa cagtaagcgaaagagccggc ctgtgagtgt aaaaacattt 4020 gaagatatcc cgttagaaga accagaagtaaaagtaatcc cagatgacaa ccagacggac 4080 agtggtatgg ttcttgcctc agaagagctgaaaactttgg aagacagaac caaattatct 4140 ccatcttttg gtggaatggt gcccagcaaaagcagggagt ctgtggcatc tgaaggctca 4200 aaccagacaa gcggctacca gtccggatatcactccgatg acacagacac caccgtgtac 4260 tccagtgagg aagcagaact tttaaagctgatagagattg gagtgcaaac cggtagcaca 4320 gcccagattc tccagcctga ctcggggaccacactgagct ctcctcctgt ttaaaaggaa 4380 gcatccacac cccaactccc ggacatcacatgagaggtct gctcagattt tgaagtgttg 4440 ttctttccac cagcaggaag tagccgcatttgattttcat ttcgacaaca gaaaaaggac 4500 ctcggactgc agggagccag tcttctaggcatatcctgga agaggcttgt gacccaagaa 4560 tgtgtctgtg tcttctccca gtgttgacctgatcctcttt tttcattcat ttaaaaagca 4620 ttatcatgcc cctgctgcgg gtctcaccatgggtttagaa caaagagctt caagcaatgg 4680 ccccatcctc aaagaagtag cagtacctggggagctgaca cttctgtaaa actagaagat 4740 aaaccaggca acgtaagtgt tcgaggtgttgaagatggga aggatttgca gggctgagtc 4800 tatccaagag gctttgttta ggacgtgggtcccaagccaa gccttaagtg tggaattcgg 4860 attgatagaa aggaagacta acgttaccttgctttggaga gtactggagc ctgcaaatgc 4920 attgtgtttg ctctggtgga ggtgggcatggggtctgttc tgaaatgtaa agggttcaga 4980 cggggtttct ggttttagaa ggttgcgtgttcttcgagtt gggctaaagt agagttcgtt 5040 gtgctgtttc tgactcctaa tgagagttccttccagaccg ttagctgtct ccttgccaag 5100 ccccaggaag aaaatgatgc agctctggctccttgtctcc caggctgatc ctttattcag 5160 aataccacaa agaaaggaca ttcagctcaaggctccctgc cgtgttgaag agttctgact 5220 gcacaaacca gcttctggtt tcttctggaatgaataccct catatctgtc ctgatgtgat 5280 atgtctgaga ctgaatgcgg gaggttcaatgtgaagctgt gtgtggtgtc aaagtttcag 5340 gaaggatttt acccttttgt tcttccccctgtccccaacc cactctcacc ccgcaaccca 5400 tcagtatttt agttatttgg cctctactccagtaaacctg attgggtttg ttcactctct 5460 gaatgattat tagccagact tcaaaattattttatagccc aaattataac atctattgta 5520 ttatttagac ttttaacata tagagctatttctactgatt tttgcccttg ttctgtcctt 5580 tttttcaaaa aagaaaatgt gttttttgtttggtaccata gtgtgaaatg ctgggaacaa 5640 tgactataag acatgctatg gcacatatatttatagtctg tttatgtaga aacaaatgta 5700 atatattaaa gccttatata taatgaactttgtactattc acattttgta tcagtattat 5760 gtagcataac aaaggtcata atgctttcagcaattgatgt cattttatta aagaacattg 5820 aaaaacttga 5830 8 19 DNAArtificial Sequence Targeting Sequence 8 tcatcacgaa gtggtgaag 19 9 21RNA Artificial Sequence Sense strand 9 ucaucacgaa guggugaagu u 21 10 21RNA Artificial Sequence Antisense strand 10 cuucaccacu ucgugaugau u 2111 21 DNA Artificial Sequence Sense strand 11 ucaucacgaa guggugaagt t 2112 21 DNA Artificial Sequence Antisense strand 12 cuucaccacu ucgugaugatt 21 13 21 DNA Artificial Sequence Targeting Sequence 13 aacgtacttgcagatgtgac a 21 14 19 DNA Artificial Sequence Targeting Sequence 14gttcatggat gtctatcag 19 15 19 DNA Artificial Sequence Targeting Sequence15 tcgagaccct ggtggacat 19 16 19 DNA Artificial Sequence TargetingSequence 16 tgacgagggc ctggagtgt 19 17 19 DNA Artificial SequenceTargeting Sequence 17 tgacgagggc ctggagtgt 19 18 19 DNA ArtificialSequence Targeting Sequence 18 catcaccatg cagattatg 19 19 19 DNAArtificial Sequence Targeting Sequence 19 acctcaccaa ggccagcac 19 20 19DNA Artificial Sequence Targeting Sequence 20 ggccagcaca taggagaga 19 2119 DNA Artificial Sequence Targeting Sequence 21 caaatgtgaa tgcagacca 1922 19 DNA Artificial Sequence Targeting Sequence 22 atgtgaatgc agaccaaag19 23 19 DNA Artificial Sequence Targeting Sequence 23 tgcagaccaaagaaagata 19 24 19 DNA Artificial Sequence Targeting Sequence 24agaaagatag agcaagaca 19 25 19 DNA Artificial Sequence Targeting Sequence25 gaaagataga gcaagacaa 19 26 19 DNA Artificial Sequence TargetingSequence 26 gatagagcaa gacaagaaa 19 27 19 DNA Artificial SequenceTargeting Sequence 27 gacaagaaaa tccctgtgg 19 28 19 DNA ArtificialSequence Targeting Sequence 28 gaaaatccct gtgggcctt 19 29 19 DNAArtificial Sequence Targeting Sequence 29 aatccctgtg ggccttgct 19 30 19DNA Artificial Sequence Targeting Sequence 30 tccctgtggg ccttgctca 19 3119 DNA Artificial Sequence Targeting Sequence 31 gcatttgttt gtacaagat 1932 19 DNA Artificial Sequence Targeting Sequence 32 gatccgcaga cgtgtaaat19 33 19 DNA Artificial Sequence Targeting Sequence 33 atgttcctgcaaaaacaca 19 34 19 DNA Artificial Sequence Targeting Sequence 34tgttcctgca aaaacacag 19 35 19 DNA Artificial Sequence Targeting Sequence35 aaacacagac tcgcgttgc 19 36 19 DNA Artificial Sequence TargetingSequence 36 aacacagact cgcgttgca 19 37 19 DNA Artificial SequenceTargeting Sequence 37 acacagactc gcgttgcaa 19 38 19 DNA ArtificialSequence Targeting Sequence 38 cacagactcg cgttgcaag 19 39 19 DNAArtificial Sequence Targeting Sequence 39 ggcgaggcag cttgagtta 19 40 19DNA Artificial Sequence Targeting Sequence 40 acgaacgtac ttgcagatg 19 4119 DNA Artificial Sequence Targeting Sequence 41 cgaacgtact tgcagatgt 1942 19 DNA Artificial Sequence Targeting Sequence 42 cgtacttgca gatgtgaca19 43 19 DNA Artificial Sequence Targeting Sequence 43 gtggtcccaggctgcaccc 19 44 19 DNA Artificial Sequence Targeting Sequence 44ggaggagggc agaatcatc 19 45 19 DNA Artificial Sequence Targeting Sequence45 gtggtgaagt tcatggatg 19 46 21 DNA Artificial Sequence TargetingSequence 46 aatcatcacg aagtggtgaa g 21 47 21 DNA Artificial SequenceTargeting Sequence 47 aagttcatgg atgtctatca g 21 48 21 DNA ArtificialSequence Targeting Sequence 48 aatcgagacc ctggtggaca t 21 49 21 DNAArtificial Sequence Targeting Sequence 49 aatgacgagg gcctggagtg t 21 5021 DNA Artificial Sequence Targeting Sequence 50 aacatcacca tgcagattat g21 51 21 DNA Artificial Sequence Targeting Sequence 51 aaacctcaccaaggccagca c 21 52 21 DNA Artificial Sequence Targeting Sequence 52aaggccagca cataggagag a 21 53 21 DNA Artificial Sequence TargetingSequence 53 aacaaatgtg aatgcagacc a 21 54 21 DNA Artificial SequenceTargeting Sequence 54 aaatgtgaat gcagaccaaa g 21 55 21 DNA ArtificialSequence Targeting Sequence 55 aatgcagacc aaagaaagat a 21 56 21 DNAArtificial Sequence Targeting Sequence 56 aaagaaagat agagcaagac a 21 5721 DNA Artificial Sequence Targeting Sequence 57 aagaaagata gagcaagaca a21 58 23 DNA Artificial Sequence Targeting Sequence 58 aagatagagcaagacaagaa aat 23 59 23 DNA Artificial Sequence Targeting Sequence 59aagacaagaa aatccctgtg ggc 23 60 23 DNA Artificial Sequence TargetingSequence 60 aagaaaatcc ctgtgggcct tgc 23 61 23 DNA Artificial SequenceTargeting Sequence 61 aatccctgtg ggccttgctc aga 23 62 23 DNA ArtificialSequence Targeting Sequence 62 aagcatttgt ttgtacaaga tcc 23 63 23 DNAArtificial Sequence Targeting Sequence 63 aagatccgca gacgtgtaaa tgt 2364 23 DNA Artificial Sequence Targeting Sequence 64 aaatgttcctgcaaaaacac aga 23 65 23 DNA Artificial Sequence Targeting Sequence 65aatgttcctg caaaaacaca gac 23 66 23 DNA Artificial Sequence TargetingSequence 66 aaaaacacag actcgcgttg caa 23 67 23 DNA Artificial SequenceTargeting Sequence 67 aaaacacaga ctcgcgttgc aag 23 68 23 DNA ArtificialSequence Targeting Sequence 68 aaacacagac tcgcgttgca agg 23 69 23 DNAArtificial Sequence Targeting Sequence 69 aacacagact cgcgttgcaa ggc 2370 23 DNA Artificial Sequence Targeting Sequence 70 aaggcgaggcagcttgagtt aaa 23 71 23 DNA Artificial Sequence Targeting Sequence 71aaacgaacgt acttgcagat gtg 23 72 23 DNA Artificial Sequence TargetingSequence 72 aacgaacgta cttgcagatg tga 23 73 23 DNA Artificial SequenceTargeting Sequence 73 aagtggtccc aggctgcacc cat 23 74 23 DNA ArtificialSequence Targeting Sequence 74 aaggaggagg gcagaatcat cac 23 75 23 DNAArtificial Sequence Targeting Sequence 75 aagtggtgaa gttcatggat gtc 2376 23 DNA Artificial Sequence Targeting Sequence 76 aaaatccctgtgggccttgc tca 23 77 21 DNA Artificial Sequence Sense strand 77accucaccaa ggccagcact t 21 78 21 DNA Artificial Sequence Antisensestrand 78 gugcuggccu uggugaggut t 21 79 18 DNA Artificial SequenceTargeting sequence 79 ggctacgtcc agcgcacc 18 80 21 DNA ArtificialSequence Targeting sequence 80 aaaccucacc aaagccagca c 21

We claim:
 1. An isolated siRNA comprising a sense RNA strand and anantisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence identical to a target sequence of about 19 to about25 contiguous nucleotides in human VEGF mRNA, human Flt-1 mRNA, or humanFlk-1/KDR mRNA, or an alternative splice form, mutant or cognatethereof.
 2. The siRNA of claim 1, wherein the human VEGF mRNA isselected from the group consisting of VEGF₁₂₁ mRNA (SEQ ID NO: 2);VEGF₁₆₅ mRNA (SEQ ID NO: 3); VEGF₁₈₉ mRNA (SEQ ID NO: 4) and VEGF₂₀₆mRNA (SEQ ID NO: 5).
 3. The siRNA of claim 1, wherein the cognate of thehuman VEGF mRNA sequence is mouse VEGF mRNA (SEQ ID NO: 1).
 4. The siRNAof claim 1, wherein the sense RNA strand comprises SEQ ID NO: 77, andthe antisense strand comprises SEQ ID NO:
 78. 5. The siRNA of claim 1,wherein the sense RNA strand comprises one RNA molecule, and theantisense RNA strand comprises one RNA molecule.
 6. The siRNA of claim1, wherein the sense and antisense RNA strands forming the RNA duplexare covalently linked by a single-stranded hairpin.
 7. The siRNA ofclaim 1, wherein the siRNA further comprises non-nucleotide material. 8.The siRNA of claim 1, wherein the sense and antisense RNA strands arestabilized against nuclease degradation.
 9. The siRNA of claim 1,further comprising a 3′ overhang.
 10. The siRNA of claim 9, wherein the3′ overhang comprises from 1 to about 6 nucleotides.
 11. The siRNA ofclaim 9, wherein the 3′ overhang comprises about 2 nucleotides.
 12. ThesiRNA of claim 5, wherein the sense RNA strand comprises a first 3′overhang, and the antisense RNA strand comprises a second 3′ overhang.13. The siRNA of claim 12, wherein the first and second 3′ overhangsseparately comprise from 1 to about 6 nucleotides.
 14. The siRNA ofclaim 13, wherein the first 3′ overhang comprises a dinucleotide and thesecond 3′ overhang comprises a dinucleotide.
 15. The siRNA of claim 14,where the dinucleotide comprising the first and second 3′ overhangs isdithymidylic acid (TT) or diuridylic acid (uu).
 16. The siRNA of claim9, wherein the 3′ overhang is stabilized against nuclease degradation.17. A recombinant plasmid comprising nucleic acid sequences forexpressing an siRNA comprising a sense RNA strand and an antisense RNAstrand, wherein the sense and an antisense RNA strands form an RNAduplex, and wherein the sense RNA strand comprises a nucleotide sequenceidentical to a target sequence of about 19 to about 25 contiguousnucleotides in human VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDRmRNA, or an alternative splice form, mutant or cognate thereof.
 18. Therecombinant plasmid of claim 17, wherein the nucleic acid sequences forexpressing the siRNA comprise an inducible or regulatable promoter. 19.The recombinant plasmid of claim 17, wherein the nucleic acid sequencesfor expressing the siRNA comprise a sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and an antisense RNA strand coding sequencein operable connection with a polyT termination sequence under thecontrol of a human U6 RNA promoter.
 20. The recombinant plasmid of claim19, wherein the plasmid is pAAVsiRNA.
 21. A recombinant viral vectorcomprising nucleic acid sequences for expressing an siRNA comprising asense RNA strand and an antisense RNA strand, wherein the sense and anantisense RNA strands form an RNA duplex, and wherein the sense RNAstrand comprises a nucleotide sequence identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in human VEGF mRNA, humanFlt-1 mRNA, or human Flk-1/KDR mRNA, or an alternative splice form,mutant or cognate thereof.
 22. The recombinant viral vector of claim 21,wherein the nucleic acid sequences for expressing the siRNA comprise aninducible or regulatable promoter.
 23. The recombinant viral vector ofclaim 21, wherein the nucleic acid sequences for expressing the siRNAcomprise a sense RNA strand coding sequence in operable connection witha polyT termination sequence under the control of a human U6 RNApromoter, and an antisense RNA strand coding sequence in operableconnection with a polyT termination sequence under the control of ahuman U6 RNA promoter.
 24. The recombinant viral vector of claim 21,wherein the recombinant viral vector is selected from the groupconsisting of an adenoviral vector, an adeno-associated viral vector, alentiviral vector, a retroviral vector, and a herpes virus vector. 25.The recombinant viral vector of claim 21, wherein the recombinant viralvector is pseudotyped with surface proteins from vesicular stomatitisvirus, rabies virus, Ebola virus, or Mokola virus.
 26. The recombinantviral vector of claim 24, wherein the recombinant viral vector comprisesan adeno-associated viral vector.
 27. A pharmaceutical compositioncomprising an siRNA and a pharmaceutically acceptable carrier, whereinthe siRNA comprises a sense RNA strand and an antisense RNA strand,wherein the sense and an antisense RNA strands form an RNA duplex, andwherein the sense RNA strand comprises a nucleotide sequence identicalto a target sequence of about 19 to about 25 contiguous nucleotides inhuman VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDR mRNA, or analternative splice form, mutant or cognate thereof.
 28. Thepharmaceutical composition of claim 27, further comprising lipofectin,lipofectamine, cellfectin, polycations, or liposomes.
 29. Apharmaceutical composition comprising the plasmid of claim 17, or aphysiologically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 30. The pharmaceutical composition of claim 29,further comprising lipofectin, lipofectamine, cellfectin, polycations,or liposomes.
 31. A pharmaceutical composition comprising the viralvector of claim 21 and a pharmaceutically acceptable carrier.
 32. Amethod of inhibiting expression of human VEGF mRNA, human Flt-1 mRNA, orhuman Flk-1/KDR mRNA, or an alternative splice form, mutant or cognatethereof, comprising administering to a subject an effective amount of ansiRNA comprising a sense RNA strand and an antisense RNA strand, whereinthe sense and an antisense RNA strands form an RNA duplex, and whereinthe sense RNA strand comprises a nucleotide sequence identical to atarget sequence of about 19 to about 25 contiguous nucleotides in humanVEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDR mRNA, or an alternativesplice form, mutant or cognate thereof, such that the human VEGF mRNA,human Flt-1 mRNA, or human Flk-1/KDR mRNA, or an alternative spliceform, mutant or cognate thereof, is degraded.
 33. The method of claim32, wherein the subject is a human being.
 34. The method of claim 32,wherein the effective amount of the siRNA is from about 1 nM to about100 nM.
 35. The method of claim 32, wherein the siRNA is administered inconjunction with a delivery reagent.
 36. The method of claim 35, whereinthe delivery agent is selected from the group consisting of lipofectin,lipofectamine, cellfectin, polycations, and liposomes.
 37. The method ofclaim 36, wherein the delivery agent is a liposome.
 38. The method claim37, wherein the liposome comprises a ligand which targets the liposometo cells at or near the site of angiogenesis.
 39. The method of claim38, wherein the ligand binds to receptors on tumor cells or vascularendothelial cells.
 40. The method of claim 39, wherein the ligandcomprises a monoclonal antibody.
 41. The method of claim 37, wherein theliposome is modified with an opsonization-inhibition moiety.
 42. Themethod of claim 41, wherein the opsonization-inhibiting moiety comprisesa PEG, PPG, or derivatives thereof.
 43. The method of claim 32, whereinthe siRNA is expressed from a recombinant plasmid
 44. The method ofclaim 32, wherein the siRNA is expressed from a recombinant viralvector.
 45. The method of claim 44, wherein the recombinant viral vectorcomprises an adenoviral vector, an adeno-associated viral vector, alentiviral vector, a retroviral vector, or a herpes virus vector. 46.The method of claim 45, wherein the recombinant viral vector ispseudotyped with surface proteins from vesicular stomatitis virus,rabies virus, Ebola virus, or Mokola virus.
 47. The method of claim 44,wherein the recombinant viral vector comprises an adeno-associated viralvector.
 48. The method of claim 32, wherein the siRNA is administered byan enteral administration route.
 49. The method of claim 48, wherein theenteral administration route is selected from the group consisting oforal, rectal, and intranasal.
 50. The method of claim 32, wherein thesiRNA is administered by a parenteral administration route.
 51. Themethod of claim 50, wherein the parenteral administration route isselected from the group consisting of intravascular administration,peri- and intra-tissue injection, subcutaneous injection or deposition,subcutaneous infusion, and direct application at or near the site ofneovascularization.
 52. The method of claim 51, wherein theintravascular administration is selected from the group consisting ofintravenous bolus injection, intravenous infusion, intra-arterial bolusinjection, intra-arterial infusion and catheter instillation into thevasculature.
 53. The method of claim 51, wherein the peri- andintra-tissue injection is selected from the group consisting ofperi-tumoral injection, intra-tumoral injection, intra-retinalinjection, and subretinal injection.
 54. The method of claim 51, whereinthe direct application at or near the site of neovascularizationcomprises application by catheter, retinal pellet, suppository, animplant comprising a porous material, an implant comprising a non-porousmaterial, or an implant comprising a gelatinous material.
 55. A methodof inhibiting angiogenesis in a subject, comprising administering to asubject an effective amount of an siRNA comprising a sense RNA strandand an antisense RNA strand, wherein the sense and an antisense RNAstrands form an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence identical to a target sequence of about 19 to about25 contiguous nucleotides in human VEGF mRNA, human Flt-1 mRNA, or humanFlk-1/KDR mRNA, or an alternative splice form, mutant or cognatethereof.
 56. The method of claim 55, wherein the angiogenesis ispathogenic.
 57. The method of claim 55, wherein the angiogenesis isnon-pathogenic.
 58. The method of claim 57, wherein the non-pathogenicangiogenesis is associated with production of fatty tissues orcholesterol production.
 59. The method of claim 57, wherein thenon-pathogenic angiogenesis comprises endometrial neovascularization.60. A method of treating an angiogenic disease in a subject, comprisingadministering to a subject in need of such treatment an effective amountof an siRNA comprising a sense RNA strand and an antisense RNA strand,wherein the sense and an antisense RNA strands form an RNA duplex, andwherein the sense RNA strand comprises a nucleotide sequence identicalto a target sequence of about 19 to about 25 contiguous nucleotides inhuman VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDR mRNA, or analternative splice form, mutant or cognate thereof, such thatangiogenesis associated with the angiogenic disease is inhibited. 61.The method of claim 60, wherein the angiogenic disease comprises a tumorassociated with a cancer.
 62. The method of claim 61, wherein the canceris selected from the group consisting of breast cancer, lung cancer,head and neck cancer, brain cancer, abdominal cancer, colon cancer,colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma,liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovariancancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm'stumor, multiple myeloma, skin cancer, lymphoma, and blood cancer. 63.The method of claim 60, wherein the angiogenic disease is selected fromthe group consisting of diabetic retinopathy, age-related maculardegeneration, and inflammatory diseases.
 64. The method of claim 63,wherein the inflammatory disease is psoriasis or rheumatoid arthritis.65. The method of claim 63, wherein the angiogenic disease isage-related macular degeneration.
 66. The method of claim 60, whereinthe siRNA is administered in combination with a pharmaceutical agent fortreating the angiogenic disease, which pharmaceutical agent is differentfrom the siRNA.
 67. The method of claim 66, wherein the angiogenicdisease is cancer, and the pharmaceutical agent comprises achemotherapeutic agent.
 68. The method of claim 66, wherein thechemotherapeutic agent is selected from the group consisting ofcisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin,daunorubicin, and tamoxifen.
 69. The method of claim 60, wherein thesiRNA is administered to a subject in combination with anothertherapeutic method designed to treat the angiogenic disease.
 70. Themethod of claim 69, wherein the angiogenic disease is cancer, and thesiRNA is administered in combination with radiation therapy,chemotherapy or surgery.